This invention relates to sialidase activity, in particular isolated sialidase enzyme and recombinant cell lines having modified sialidase activity.
Sialidases are a family of glycohydrolytic enzymes which cleave sialic acid residues from the oligosaccharide components of glycoproteins and glycolipids. Viral and bacterial enzymes have been studied, for example the influenza sialidases in particular (Air, G. M. and Laver, W. G. (1989) Proteins: Struct. Func. Genet., 6:341), but mammalian sialidases have not been well characterized. For the most part, studies of mammalian sialidases have been confined to investigation of substrate specificities and kinetic analysis using partially purified preparations, although a sialidase from rat liver and muscle has been purified to homogeneity (Miyagi, T. and Tsuiki, S. (1985) J. Biol. Chem., 260:6710). Sialidases have been identified in a number of cellular organelles: the plasma membrane (Schengrund, C., Rosenberg, A., and Repman, M. A. (1976) J. Cell. Biol., 70:555), the lysosomes and the cytosol (Tulsiani, D. R. P., and Carubelli, R., (1970) J. Biol. Chem., 245:1821).
Glycoproteins are often produced by expression of encoding genes in recombinant host cells in vitro, the cells having the normal enzyme components of cellular glycosylation machinery. Sialic acid in the oligosaccharide component of a glycoprotein is involved in mediation of clearance from the serum and affects the physical, chemical and immunogenic properties of the protein molecule. It is therefore important to maintain the sialic acid content of glycoproteins, particularly of those proteins intended for use as therapeutics.
The present invention is based on modification in a recombinant cell line of the constitutive expression of genes which encode enzymes which are involved in the destruction or production of the oligosaccharide portions of glycoproteins. In particular, the modification to the recombinant cell line may be such as to ensure that the gene or genes of interest are not functionally expressed. Of particular interest is a sialidase gene within a recombinant cell line, especially a gene encoding a cytosolic sialidase. Example cell lines are those derived from Chinese hamster ovaries and human embryonic kidneys.
In the recombinant cell line functional gene expression may be disrupted by mutation, addition or deletion of one or more nucleotides. Such mutation, addition or deletion may be by any of the methods known to the person skilled in the art, for instance homologous recombination between the genomic gene and a differing but largely homologous nucleic acid sequence introduced into the cells. The gene may be deleted altogether.
The gene may be not functionally expressed by virtue of disruption of the gene function by regulation of its transcription or translation, eg using antisense RNA.
The present invention also provides a substantially homogeneous sialidase which can be obtained from cell culture fluid of a Chinese hamster ovary cell line. Characteristics of such a sialidase are described and discussed infra.
It also provides an oligonucleotide probe which is useful in obtaining a sialidase-encoding gene and a nucleic acid sequence obtained by a process comprising hybridizing the probe with nucleic acid in a mammalian DNA library to form hybrids which can be isolated. The nucleic acid may be used for expression of sialidase. It may be modified in all manner of ways, eg by mutation, addition or deletion of one or more nucleotides, amplification, cleavage and tailoring.
In a preferred embodiment of the present invention a sialidase gene of a cell line is disrupted so that it is not functionally expressed, the level of functional sialidase produced by the cells being such that sialic acid residues in the carbohydrate side-chains of glycoprotein produced by the cells are not cleaved, or are not cleaved to an extent which affects the function of the glycoprotein. Such cells are useful as host cells for the expression of recombinant glycoproteins from nucleic acid transformed into the cells under appropriate conditions. Glycoproteins produced by expression of encoding nucleic acid introduced into these cells should have intact, functional carbohydrate side chains.
Recombinant Cell Line
This expression refers to cells established in ex vivo culture and which have some genetic modification from the original parent cells from which they are derived. Such genetic modification may be the result of introduction of a heterologous gene for expression of the gene product, or it may be by the introduction of a gene, possibly with promoter elements, for production within the cells of antisense RNA to regulate expression of another gene. Equally, the genetic modification may be the result of mutation, addition or deletion of one or more nucleotides of a gene or even deletion of a gene altogether, by any mechanism. Cells of a recombinant cell line used in the production of a desired protein product have the means for glycosylating proteins by addition of oligosaccharide side chains. Such cells also have the capability to remove and/or modify enzymatically part or all of the oligosaccharide side chains of glycoproteins.
Functional Expression, and Grammatically Related Terms
Functional expression of a gene refers to production of the protein product encoded by the gene in a form or to the extent required for the product to perform its normal function within the cell environment. Thus, a gene encoding an enzyme involved in protein glycosylation, or deglycosylation, is functionally expressed when enough of the enzyme is produced in a working form to glycosylate, or deglycosylate, at a normal level protein produced in the cell. Functional expression of a gene may be disrupted by modification of the nucleotide sequence of the gene so that protein product of the gene is defective in its function, or by deletion or modification of part or all of promoter sequences associated with the gene and involved in transcription of the gene, or by deletion of the gene itself from the genome of the cell, or by interference with translation of mRNA transcribed from the gene eg interference by antisense RNA, or by any combination of any of these with each other or with any other means known to the person skilled in the art for disrupting gene function.
The terms xe2x80x9cDNA sequence encodingxe2x80x9d, xe2x80x9cDNA encodingxe2x80x9d and xe2x80x9cnucleic acid encodingxe2x80x9d refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. The DNA sequence thus codes for the amino acid sequence.
The terms xe2x80x9creplicable expression vectorxe2x80x9d and xe2x80x9cexpression vectorxe2x80x9d refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its insert (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized.
The terms xe2x80x9ctransformed host cellxe2x80x9d and xe2x80x9ctransformedxe2x80x9d refer to the introduction of DNA into a cell. The cell is termed a xe2x80x9chost cellxe2x80x9d, and it may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are mammalian, such as Chinese hamster ovary cells or human embryonic kidney 293 cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the same species as the host cell or a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.
xe2x80x9cDigestionxe2x80x9d, xe2x80x9ccuttingxe2x80x9d or xe2x80x9ccleavingxe2x80x9d of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site. Restriction enzymes are commercially available and are used according to the instructions supplied by the manufacturers. Restriction enzymes are designated by abbreviations composed of a capital letter followed by two or three lower case letters representing the microorganism from which each restriction enzyme was obtained. These letters are followed by one or more Roman numerals that identify the particular enzyme. In general, about 1 xcexcg of plasmid or DNA fragment is used with about 2 unites of enzyme in about 20 xcexcl of buffer solution. The appropriate buffer, substrate concentration, incubation temperature, and incubation time for each enzyme is specified by the manufacturer. After incubation, the enzyme and other contaminants are removed from the DNA by extraction with a solution of phenol-chloroform, and the digested DNA is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme may be followed by treatment with bacterial alkaline phosphatase or calf intestinal alkaline phosphatase. This prevents the two restriction cleaved ends of a DNA fragment from xe2x80x9ccircularizingxe2x80x9d or forming a closed loop that would impede insertion of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not followed by 5xe2x80x2 terminal dephosphorylation. These procedures and reagents for dephosphorylation are described in sections 1.60-1.61 and sections 3.38-3.39 of Sambrook et al., (Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, New York [1989]).
xe2x80x9cRecoveryxe2x80x9d or xe2x80x9cisolationxe2x80x9d of a given fragment of DNA from a restriction digest means separation of the resulting DNA fragment on a polyacrylamide or an agarose cell by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example see R. Lawn et al., 1981, Nucleic Acids Res., 9:6103, and D. Goeddel et al., 1980, Nucleic Acids Res., 8:4057.
xe2x80x9cSouthern Analysisxe2x80x9d or xe2x80x9cSouthern blottingxe2x80x9d is a method by which the presence of DNA sequences in a digest or DNA-containing composition is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment. Southern analysis refers to the separation of digested DNA on an agarose gel, denaturation of the DNA, and transfer of the DNA from the gel to a nitrocellulose or nylon membrane using methods originally described by Southern (J. Mol. Biol., 98:503 [1975]) and modified as described in sections 9.31-9.57 of Sambrook et al., supra.
xe2x80x9cTransformationxe2x80x9d means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or chromosomal integrant. The method used for transformation depends on whether the host cell is a eukaryote or a prokaryote. A preferred method used to transform prokaryotes is the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Eukaryotes may be transformed using the calcium phosphate method as described in sections 16.32-16.37 of Sambrook et al., supra.
xe2x80x9cLigationxe2x80x9d refers to the process of forming phosphodiester bonds between two double stranded DNA fragments using the enzyme ligase in a suitable buffer that also contains ATP.
xe2x80x9cOligonucleotidexe2x80x9d refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds. The oligonucleotides may be chemically synthesized by known methods and purified on polyacrylamide gels.
Neu5Ac2en, 5-acetamide-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid; 9-azido-Neu5Ac2en, 5-acetamido-2,6-anhydro-9-azido-3,5,9-trideoxy-D-glycero-D-galacto-non-2-enonic acid; 9-PANP-Neu5Ac2en, 9-S-(4xe2x80x2-azido-2xe2x80x2-nitro-phenyl)-5-acetamido-2,6 anhydro-9-thio-2,5,9-trideoxy-D-glycero-D-galacto-non-2-enonic acid; 4-MU-Neu5Ac, (4-methylumbelliferyl-5-acetamido-3,5-dideoxy- D-glycero-xcex1-D-galacto-nonulopyranosid)onic acid; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; CHO, Chinese hamster ovary; EDTA, ethylene diamine tetraacetic acid; DEAE; diethylaminoethyl-; GM1, ||3NeuAc-GgOse4Cer; GM2, ||3NeuAc-GgOse3Cer; GM3, ||NeuAc-LacCer; GD1a, IV3NeuAc, ||3NeuAc-GgOse4Cer; GD1b, ||3(NeuAc)2-GgOse4Cer; GT1b, IV3NeuAc, ||3(NeuAc)2-GgOse4Cer.
Amino acids are designated thus: